Microtubule (MT) cytoskeleton provides an important control-point of endothelial barrier regulation; however, the role of this key cytoskeleton element has not been well studied. The MT stabilizing drug taxol has been shown to attenuate the lung injury in mice models suggesting that MTs may be important in mediating increased lung vascular permeability. However, taxol displays a general toxicity that makes it an inconvenient drug for doctors and their patients.
Microtubule end binding proteins are highly conserved microtubule plus-end tracking accessory factors that bind to growing microtubules (MTs) and suppress MT catastrophic events. Two such end binding proteins, EB1 and EB3, play roles in regulating endothelial cytoskeletal dynamics and cell shape change, the primary determinants of the permeability of endothelial barrier.
Ca2+ is a highly versatile second messenger that regulates endothelial permeability and vascular homeostasis. The activation of phospholipase C β (PLCβ), downstream of PAR-1 activation mediates hydrolysis of phosphotidyl inositolbisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates Ca2+ release from IP3-sensitive intracellular stores, i.e., the endoplasmic reticulum (ER). The depletion of Ca2+ from ER stores is mediated by activation of IP3R on the ER membrane and leads to transient increase in intracellular Ca2+. Ca2+ entry or “influx” is mediated by transient receptor potential canonical (TRPC) channels that are permeable to various cations including Ca2+ and Mg2+. TRPC1 and 4 are store-operated Ca2+ channels (SOC) in endothelial lung microvascular cells that are activated by depletion of ER.
The increase in intracellular concentration of Ca2+ up-regulates activity of protein kinase Cα (PKC α). PKC α is a key regulator of the endothelial permeability response to multiple mediators 2. PKC α phosphorylates p120-catenin and mediates its dissociation from VE-cadherin, thus resulting in VE-cadherin internalization. PKC α also acts upstream of RhoA activation by phosphorylating p115RhoGEF and GDI-1. RhoA in turn facilitates phosphorylation-induced inhibition of myosin light chain phosphatase (MLCP) by activating Rho kinase (ROCK). The inhibition of MLCP is accompanied by the Ca2+/calmodulin-dependent activation of MLCK that leads to phosphorylation of MLC and induces acto-myosin contraction in response to pro-inflammatory mediators such as thrombin and histamine.
The integrity of MT cytoskeleton is required for IP3-induced Ca2+ release from ER stores. Alteration of MT dynamics by MT destabilizing or MT stabilizing agents nocodazole, colchicine and taxol inhibits IP3-gated release of Ca2+, suggesting that MT dynamics are required for full activation of IP3R. The MT cytoskeleton is involved in remodeling of ER, thus ensuring organization and propagation of Ca2+ waves in response to external stimuli. The ER attaches to and elongates together with MT growing ends though direct interaction of EB1 and EB3 with stromal interaction molecule 1 (STIM1). Depletion of EB1 in HeLa (HeLa cells do not express EB3) decreases ER protrusion events, however does not inhibit activation of SOC by thapsigargin suggesting that some other mechanisms are involved in activation of SOC and propogation of calcium signaling in epithelial cells. In endothelial cells, the localization of IP3R in caveolae is critical for both ER Ca2+ store depletion and SOC activation. This indicates that activation of IP3R and/or its responsiveness to IP3 is important element of calcium signaling. It is proposed that MT cytoskeleton positively regulates IP3R activation in response to IP3 and thus transmits extracellular signals throughout the cell, eliciting a physiological response.
Despite the advanced supportive care in the United States, about 100,000 patients die annually from Acute Lung Injury (ALI), a complex inflammatory response associated with neutrophil infiltration and release of cytokine and pro-inflammatory mediators during sepsis. Hyper-permeability of lung vessels, a characteristic feature of the disease, leads to the formation of pulmonary edema. Novel therapies are needed to prevent or treat dysfunction of endothelial barrier.